p21 Protects Against Fluoride Toxicity in LS8 Cells

Objectives: Fluoride overexposure is an environmental health hazard and can cause enamel and skeletal fluorosis. Previously we demonstrated that fluoride increased acetylated-p53 and its downstream target p21 in Ameloblast-derived LS8 cells. p21 can trigger cell cycle arrest or apoptosis, while phospho-p21 (p-p21) inhibits apoptosis. MDM2 (ubiquitin E3 ligase) is a p53 target and MDM2 suppresses the p53 pathway through a negative feedback mechanism. MDM2 binds to p53 or p21 and promotes p53 or p21 proteasomal degradation. However, MDM2 function in fluoride toxicity remains obscure. This study assessed MDM2 function in fluoride toxicity.
Methods: LS8 cells were treated with NaF with/without MG132 (proteasome inhibitor) or Nutlin-3a (MDM2 antagonist). p53, MDM2, p21 expression and apoptosis were assessed by qPCR, western blot, immunocytochemistry, and co-immunoprecipitation. C57BL/6 mice were treated with fluoride-containing water to evaluate, by use of immunohistochemistry, the level of phospho-MDM2 (p-MDM2) which causes p53 and p21 proteasome-mediated degradation in enamel organ.
Results: NaF treatment for 2 to 6 h increased p-p21 levels which can inhibit apoptosis. However, p-p21 and p21 were decreased by NaF at 24 h, even though p21 mRNA was significantly increased at 24 h. Proteasome inhibitor MG132 reversed the fluoride-mediated p21 decrease, indicating that fluoride facilitates p21 proteasomal degradation. MG132 suppressed fluoride-induced caspase-3 cleavage, suggesting that the proteasome plays a pro-apoptotic role in fluoride toxicity. NaF increased Mdm2 mRNA in vitro and increased p-MDM2 levels both in vitro and in vivo. NaF increased MDM2-p21 binding and p53-MDM2 binding and this was suppressed by treatment with the MDM2 antagonist Nutlin-3a. Nutlin-3a reversed p21 attenuation, and increased p-p21 to suppress apoptosis after 24 h NaF treatment. These results suggest that MDM2-mediated p21 proteasomal degradation with subsequent p-p21 attenuation contributes to fluoride-induced apoptosis.
Conclusions: Inhibition of MDM2-mediated p21 degradation may be a potential therapeutic target to mitigate fluoride toxicity.